Naturally developing tissue is intrinsically of a multi-cell-type nature. A substantial portion of cultured stem cells that are implanted, die without reaching maturity or integrating themselves into a functional tissue system. The odds of survival and functional integration increase when cultured cells are allowed to develop along side of their natural companion cells. In many cases, the number of surviving cells may be improved by growing glial cells and endothelial cells or fibroblasts along with neurons. This generally holds true both in culture, and after implantation.
Tissue culture, involving the growth of tissues and/or cells separate from the organism, is typically facilitated by use of a liquid, semi-solid, or solid growth media, such as broth or agar. When intended for implantation as a solid organ, e.g., in the context of regenerative medicine, a suitable matrix is usually required. Even with the appropriate immature cells (e.g., stem cells) in place, development into function, and/or implantable tissue does not occur spontaneously. In the specific case of neural tissue, for example, brain, axonal and dendritic sprouting is shaped by activity of the various cells in the milieu. In this way, local cellular environments are crucial in the regulation of neurogenesis. Empirically, scientists have evidenced that hippocampal cell co-culture promotes hippocampal neurogenesis, and that adult NPCs grown in an environment non-permissive for neurogenesis are unable to respond to excitation. These cells communicate with one another, e.g., via chemical, molecular and electrical signals. Frequently, chemical or molecular signaling is triggered by electrical signaling; for example an endocrine cell releasing a growth factor when electrically stimulated. Activity-dependent competition frequently occurs in this context. For example, more active neurons from one brain region may overgrow regions occupied by less active neurons. Conversely, limiting activity in a brain region during development results in functional deficits. Electrical signaling and molecular signaling are the most common approaches by which cells in culture control mutual behavior within the milieu.
Electrical signaling is an important part of nerve cell development and for many other types of cells including endocrine cells and muscle cells. The application of electrical pulses to neuronal progenitor cells (NPCs) causes them to evolve from generic sphere-like structures into mature neurons, sprouting axons and dendrites along the way, and establishing electrical connections with other neurons.
Chemical/molecular signaling is frequently triggered by electrical signaling. For example, adult neurogenesis and maturation of NPCs is greatly enhanced by excitatory stimuli and involves Cav1.2/1.3 channels and NMDA receptors. These Ca2+ influx pathways are located on the proliferating NPCs, allowing them to directly sense and process excitatory stimuli. The Ca2+ signal in NPCs leads to rapid induction of a gene expression pattern that facilitated neural development. This leads to synaptic incorporation of new neurons into active neural circuits. Another example is endocrine cell releasing a growth factor when electrically stimulated, but may also be triggered by other molecular or chemical signals. Nerve growth factor (NGF) is secreted by cells surrounding a developing neuron, such as glial cells, and is critical to the development and long-term survival of neurons. Nerve growth factor (NGF), is a small protein secreted by glial cells as well as by some neurons, and induces the differentiation and survival of target neurons. NGF binds to and activates its high affinity receptor (TrkA), and a low-affinity receptor (LNGFR), and promotes neuron survival and differentiation. Conversely, molecular modifications of NGF such as proNGF can elicit apoptosis. Brain-derived neurotrophic factor (BDNF) is released from cells including fibroblasts and endothelial cells (such as those within capillaries), and serves to promote growth and development of neurons, including axonal and dentdritic sprouting. Deficient expression of BDNF not only impairs the development of neurons, but also impairs the development of capillaries and the survival of endothelial cells themselves. NGF, BDNF and neurotrophin-3 bind to the neurons bearing tyrosine kinase (trk) receptors trk A, trk B and trk C. Vascular endothelial growth factor (VEGF)-D is a member of the VEGF family of angiogenic growth factors that recognizes and activates the vascular endothelial growth factor receptor (VEGFR)-2 and VEGFR-3 on blood and/or lymphatic vessels. Neuropilin-1 (NRP-1), for example, is one of the vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) receptors that is involved in normal vascular development.
Electrical and chemical/molecular signaling has limitations, however. For example, electrical stimulation is rather agnostic to the types of cells that it activates. In brief, an electric field of a given distribution displays relatively low preference with respect to the type of cells which they affect. Electrodes indiscriminately influence the behavior of activate neurons, glia, endocrine cells, muscle cells, and even the growth of bone within the stimulated area. As a result, physical proximity of an electrode pole to a given cell may be the single largest determining factor as to whether or not it is affected. Because of these limitations, it is generally not possible to exclusively affect a specific class of cell in heterogeneously populated tissue.
Intercellular molecular signaling, although frequently cell-type specific, is often not readily modified artificially in a physically tightly knit cell culture environment, which frequently resists permeation of required growth factors, particularly in the absence of efficient capillary development. Proper and/or ideal distribution of chemical and molecular signaling agents including K+, BDNF, NGF, and VEGF may be best achieved using the cells that natively produce these agents, in their natural spatial configurations with respect to the target cells. Because molecular signaling is frequently triggered by electric signals to the source cell, such signaling is subject to the non-specify of electrical activity within the milieu.
There are a number of challenges to successful production of a cultured neuronal tract using stem cells (either adult stem cells or embryonic stem cells). These challenges have included issues emanating from maturing stem cell arrays remaining in evolution continuously, and connections being made between them early in their life where the connections may or may not be maintained as they develop further. Some method of ongoing functional reinforcement, either natural or artificial, is likely necessary for the long term viability of a cultured tract.
Efforts continue toward the goal of facilitating the consistent sprouting and growth of dendrites and axons in a predictable direction, as present studies show their natural development tendency to be lateral and/or randomly-directed growth.